Insulin mimotopes and methods of using the same

ABSTRACT

Methods for inhibiting an autoimmune disease by administering to a subject a therapeutically effective amount of a composition that induces conversion of naive T cells into Foxp3+ regulatory T cells to induce immunosuppression in the subject. Methods for detecting in a subject an autoimmune disease or a predisposition to an autoimmune disease, and methods for assessing the efficacy of a therapy for an autoimmune disease, particularly type 1 diabetes.

GOVERNMENT INTEREST

This invention was made with Government support under grant numbers R01DK032083 and K08 DK095995 awarded by the National Institute of Diabetesand Digestive Kidney Diseases. The U.S. Government has certain rights inthe invention.

TECHNICAL FIELD

The invention relates to the field of autoimmune disorders, specificallyto the monitoring and treatment of diabetes.

BACKGROUND

Type 1 diabetes (T1D), the autoimmune form of diabetes, results from Tcell mediated destruction of insulin producing beta cells withinpancreatic islets (1). The disease is dramatically increasing inincidence, doubling in the last two decades, and is predictable by themeasurement of antibodies directed against proteins in beta cells (2-4).Despite being predictable, T1D onset cannot be delayed or prevented.Major efforts at disease prevention have been undertaken usingpreparations of insulin (subcutaneous, oral, and intranasal) to inducetolerance and delay the onset of clinical symptoms (5-7). Measuringinsulin-specific T cell responses from the peripheral blood has been achallenging feat, but would allow for assessment of therapeutic response(e.g. converting an inflammatory T cell response (Th1) into a regulatoryresponse), which has been a major obstacle in these trials.

Thus, there exists a need for improved methods of identifying andmonitoring T1D-associated T cell responses in individuals, to select andadminister individualized therapies to prevent or treat the disease andto efficiently and effectively monitor T1D disease progression aftertherapies are administered.

SUMMARY

Insulin is a major self-antigen for both T and B cells in murine andhuman T1D with insulin B chain amino acids 9-23 (B:9-23), a key epitopepresented by major histocompatibility (MHC) class II molecules to CD4 Tcells targeting pancreatic beta cells (8-10). There is strong evidencefrom the nonobese diabetic (NOD) mouse model of spontaneous autoimmunediabetes that pathogenic CD4 T cells recognize insulin B:9-23 presentedin an unfavorable binding position or ‘register’ by the NOD MHC class IImolecule, IA^(g7) (9, 11, 12). A unique polymorphism in the IA^(g7) betachain at position 57 (Asp->Ser) favors the binding of peptides thatplace an acidic amino at the p9 position of its peptide binding groove.The B22 Arg of B:9-23 is a very poor match for this pocket when thepeptide is bound in the pathogenic register. The binding of the peptidein this register can be greatly enhanced by creating a mimotope withmutation of B22 Arg→Glu, which now places the highly favorable acidicGlu in the p9 pocket. This peptide mimotope stimulates B:9-23 specificCD4 T cells about 100-fold better than the wild type peptide andfluorescent IA^(g7) tetramers made with the altered peptide detect CD4 Tcells in the pancreas and pancreatic lymph nodes of prediabetic NOD mice(12). In addition, this mimotope, but not the wild type B:9-23 peptide,administered at low doses, is capable of inducing tolerance andcompletely preventing diabetes onset in the NOD mouse (13).

Similar to the NOD class II molecule, human MHC class II genes, termedhuman leukocyte antigen (HLA), explain more than 50% of the genetic riskfor T1D (14). The HLA-DQ8 (DQB*03:02) and DQ2 (DQB*02:01 and DQB*02:02)alleles increase risk for disease development with approximately 90% ofall T1D individuals having one or both alleles (14-16). Strikingly, thepolymorphic HLA-DQ6 (DQB*06:02) allele provides dominant protection fromdiabetes development (14).

The inventors sought to detect peripheral T cell responses to insulin innew-onset T1D patients, longstanding T1D patients and non-diabeticcontrols utilizing novel, modified insulin B chain peptides. Because thebeta chains of DQ2 and DQ8 also bear a unique polymorphism at position57 that favors peptides with acidic amino acids at the p9 position oftheir binding grooves (17), the inventors hypothesized that diabetogenicinsulin reactive CD4 T cells in T1D patients may also recognize B:9-23bound to DQ2 or DQ8 in the unfavorable register. If so, the B:9-23mimotope with the B22 Arg→Glu mutation might detect these T cells muchbetter than the wild type peptide. Therefore, the inventors examined Tcells in the peripheral blood of new-onset T1D individuals for theirresponses to the mimotope vs. wild type B:9-23 peptide, and foundnumerous new-onset T1D patients with a robust inflammatory IFN-γresponse to the insulin B:9-23 mimotope, but not the wild type peptide(5). In contrast, control subjects without diabetes or isletautoantibodies produced a regulatory Interleukin-10 (IL10; also known ashuman cytokine synthesis inhibitory factor (CSIF) response to theinsulin mimotope only if a diabetes protective allele was present,suggesting the presence of T cell tolerance to insulin in theseindividuals. The T cell responses were DQ restricted in T1D subjectswith established disease as proliferation of CD4 T cells to the insulinmimotope could be blocked by a DQ monoclonal antibody. Analysis of Tcell receptor V gene usage in the proliferating cells demonstratesskewing and clustering of dominantly used V alpha genes based uponsimilarity in complementarity determining regions (CDRs).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a flowchart of the subjects and insulin peptides used forcytokine ELISPOT assays. The top of FIG. 1 shows a flowchart of thepatients enrolled into the study with subgroups based upon disease andβ57 aspartic acid-containing HLA-DQ alleles, and the bottom of FIG. 1shows an amino acid sequence of the native insulin B:9-23 peptide andmimotopes and substitutions. The amino acids predicted to anchor eachpeptide to the DQ peptide binding groove in a low-affinity register ofbinding are shown in subscript. Thus, the B22 arginine is an unfavorablematch for pocket 9 in the MHC groove. The two mimotopes shown in FIG. 1have identical amino acid substitutions at position 8 and 9 to thosewhich bind the NOD class II molecule, I-A^(g7), and activate insulinspecific T cells.

FIG. 2 is a comparison of IFN-γ to IL10 ELISPOT responses in T1D andcontrol subjects to the insulin B:9-23 (B22E) mimotope. Each dotrepresents a single individual having both cytokines measured. Despiteproducing IFN-γ, controls make robust IL10 responses to the insulinmimotope. Controls with at least one β57Asp DQ allele (black and lightgrey circles) are IL10 responders, as 17/18 (94%) make more than 5 IL10spots compared to 3/8 (38%) with no β57Asp DQ alleles (dark greycircles), p=0.005.

FIG. 3 shows IL10 ELISPOT responses in T1D and control subjects. Asshown on the left, independent of DQ genotype, controls have a greaterIL10 response to the native insulin B:9-23 peptide and a trend towardsmore with the insulin B22E mimotope compared to T1D. As shown on theright, by analyzing just control subjects, those with at one DQ allele(second column open circles) having the protective β57 aspartic acidpolymorphism (n=18) produce greater IL10 responses to the insulin B22Emimotope than control subjects with two non-β57Asp DQ alleles (secondcolumn, solid circles) (n=9). Each dot represents the total number ofIL10 ELISPOTs from 106 PBMCs for a single individual. The mean IL10background response in the control subjects was 3.0 spots.

FIG. 4A shows the proliferation of unfractionated PBMCs with insulinpeptides from longstanding T1D subjects with two non-β57Asp DQ alleles.Isolated and unfractionated PBMCs were labeled with CFSE, culturedwithout any in vitro stimulus (no cytokines, anti-CD3 or anti-CD28antibodies) other than peptide, and analyzed by flow cytometry for CFSEdilution and cell surface markers after 7 days of culture. FIG. 4A showsrepresentative data from two T1D subjects with CFSE proliferationassays. CD4 T cells proliferate in response to the B22E mimotope withoutthe in vitro addition of cytokines. PBMCs labeled with CFSE (no antigenstimulus in culture) are a negative control and Pentacel (childhoodvaccine containing 5 different immunogens) is a positive control. FIG.4B shows summary data of proliferative responses comparing CFSE only (noantigen background) to the B22E mimotope, and FIG. 4C shows aproliferation of native insulin B:9-23 to the mimotope. FIG. 4D shows aninhibition curve of a DQ antibody blocking CD4 T cell proliferation froma T1D with two non-β57Asp DQ alleles (DQ8/8 homozygote). Antibody wasadded in culture with CFSE labeled PBMCs and B22E mimotope for theentire 7-day culture period. Percentage of inhibition was calculatedfrom proliferation of CD4+CFSElo cells to the insulin B22E mimotope.FIG. 4E shows summative data of proliferative responses to the mimotopewith and without DQ antibody.

FIGS. 5A-5C show T cell receptor (TCR) V gene skewing afterproliferation to the insulin B22E mimotope. FIG. 5A is summative datafor Vα gene sequencing before and after stimulation from three T1Dsubjects all having two non-β57Asp DQ alleles. TCR alpha chain geneswere sequenced to identify V gene usage (TRAV and TRDV) from CD4+ cellsprior to proliferation and then on sorted CD4+CFSElo cells afterproliferation to the insulin B22E mimotope. Data are depicted as thefold change (proliferated/baseline) for each V gene and show the mean+/−SEM. FIG. 5B is a phylogenetic tree of V genes based upon similarity inCDR1 and (FIG. 5C) CDR2 regions. Four predominant V genes (grey text) inthe proliferated cells of all 3 patients cluster together based uponsimilarity in CDR1 and CDR2 sequences.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is drawn to mimotopes of insulin peptides andmethods of using the same to induce immunosuppression, prevent diabetesonset and monitor disease progression and treatment regimens.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. In case of conflict,the present specification, including explanations of terms, willcontrol. The materials, methods and examples are illustrative only andnot intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Non-limiting examples include complete Freund's adjuvant(CFA), incomplete Freund's adjuvant (IFA), aluminum salts, Amplivax (CpGoligodeoxynucleotides; Mosemann et al., J. Immunol. 173:4433, 2004), andIVX-908 (ID Biomedical of Canada). Development of vaccine adjuvants foruse in humans is reviewed in, for example, Singh et al. (Nat.Biotechnol. 17:1075-1081, 1999).

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” or “patient”includes both human and veterinary subjects, for example, humans,non-human primates, dogs, cats, horses, and cows.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

Autoimmune Disease: A disease in which the immune system produces animmune response (for example, a B cell or a T cell response) against anantigen that is part of the normal host (that is, an autoantigen), withconsequent injury to tissues. An autoantigen may be derived from a hostcell, or may be derived from a commensal organism such as themicro-organisms (known as commensal organisms) that normally colonizemucosal surfaces. An exemplary autoimmune diseases affecting humansincludes type 1 diabetes (T1D).

Beta interferon: Any beta interferon including interferon-beta 1a andinterferon-beta 1b. Interferon-beta 1a is a 166 amino acid glycoproteinwith a predicted molecular weight of approximately 22,500 daltons. Theinterferon beta 1a known as Avonex® is produced by recombinant DNAtechnology utilizing mammalian cells (Chinese Hamster Ovary cells) intowhich the human interferon-beta gene has been introduced. The amino acidsequence of Avonex® is identical to that of natural humaninterferon-beta.

Cluster of differentiation factor 4 (CD 4) is a T-cell surface proteinthat mediates interaction with MHC class II molecules. CD4 is a 55 kDatransmembrane glycoprotein belonging to the immunoglobulin superfamily.A T-cell that expresses CD4 is a “CD4⁺” T-cell. Likewise, a T-cell thatdoes not express CD4 is a “CD4⁻” T-cell.

Cluster of differentiation factor 25 (CD 25), the IL-2 receptor alphachain. A T cell that expresses CD25 is a “CD25+” T cell.

Cytokine: The term “cytokine” is used as a generic name for a diversegroup of soluble proteins and peptides that act as humoral regulators atnano- to picomolar concentrations and which, either under normal orpathological conditions, modulate the functional activities ofindividual cells and tissues. These proteins also mediate interactionsbetween cells directly and regulate processes taking place in theextracellular environment. Many cytokines act as cellular survivalfactors by preventing programmed cell death. Cytokines include bothnaturally occurring peptides and variants that retain full or partialbiological activity.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response for example, an epithelial cell that secretesinterferon or a cytokine. An immune response includes, but is notlimited to, an innate immune response or inflammation.

Immunosuppression: Nonspecific unresponsiveness of cellular and/orhumoral immunity. Immunosuppression refers to the prevention ordiminution of an immune response and occurs when T and/or B cells aredepleted in number or suppressed in their reactivity, expansion ordifferentiation. Immunosuppression may arise from activation of specificor non-specific Treg cells, from cytokine signaling, in response toirradiation, or by drugs that have generalized immunosuppressive effectson T and B cells.

Immunosuppressive agent: A molecule, such as a chemical compound, smallmolecule, steroid, nucleic acid molecule, or other biological agent,that can decrease an immune response such as an inflammatory reaction.Immunosuppressive agents include, but are not limited to an agent of usein treating an autoimmune disorder. Specific, non-limiting examples ofimmunosuppressive agents are non-steroidal anti-inflammatory agents,cyclosporine A, and anti-CD4 antibodies.

Inflammation: A complex series of events, including dilatation ofarterioles, capillaries and venules, with increased permeability andblood flow, exudation of fluids, including plasma proteins andleucocytic migration into the inflammatory focus.

Inflammation may be measured by many methods well known in the art, suchas the number of leukocytes, the number of polymorphonuclear neutrophils(PMN), a measure of the degree of PMN activation, such as luminalenhanced-chemiluminescence, or a measure of the amount of cytokinespresent.

Inhibiting or Treating a Disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as an autoimmune disease (e.g., T1D). “Treatment” refers toa therapeutic intervention that ameliorates a sign or symptom of adisease or pathological condition after it has begun to develop. As usedherein, the term “ameliorating,” with reference to a disease orpathological condition, refers to any observable beneficial effect ofthe treatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease.

Isolated/purified: An “isolated” or “purified” biological component(such as a nucleic acid, peptide or protein) has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, that is, other chromosomal and extrachromosomal DNA and RNA, andproteins. Nucleic acids, peptides and proteins that have been “isolated”thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids or proteins. The term “isolated” or“purified” does not require absolute purity; rather, it is intended as arelative term. Thus, for example, an isolated biological component isone in which the biological component is more enriched than thebiological component is in its natural environment within a cell.Preferably, a preparation is purified such that the biological componentrepresents at least 50%, such as at least 70%, at least 90%, at least95%, or greater of the total biological component content of thepreparation.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are fivemain types of leukocytes, subdivided into two main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes).

Lymphocyte: Any of the mononuclear nonphagocytic leukocytes, found inthe blood, lymph, and lymphoid tissues (such as the thymus), that arethe body's immunologically competent cells and their precursors.Lymphocytes are divided on the basis of ontogeny and function into atleast two classes, B and T lymphocytes (a.k.a., B and T cells), whichare responsible for humoral and cellular immunity, respectively.

Peptide: A polymer in which the monomers are amino acid residues whichare joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “peptide” or “polypeptide” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “peptide” is specificallyintended to cover naturally occurring peptides, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a peptide, polypeptide, or protein.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in the methods disclosed herein are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of TCR peptides andadditional pharmaceutical agents. In general, the nature of the carrierwill depend on the particular mode of administration being employed. Forinstance, parenteral formulations usually comprise injectable fluidsthat include pharmaceutically and physiologically acceptable fluids suchas water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions(e.g., powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, salts, amino acids, and pHbuffering agents and the like, for example sodium or potassium chlorideor phosphate, Tween, sodium acetate or sorbitan monolaurate.

Pulsatile Dose: A dose administered as a bolus. A pulsatile dose can beadministered to a subject as a single administration, such as by directinjection or by an intravenous infusion during a specified time period.Thus, the pulsatile dose can be a “push” or rapid dose, but need not be,as it can be administered over a defined time period, such as in aninfusion. Repeated pulsatile doses can be administered to a subject,such as a bolus administered repeatedly, such as about every one, two,or three months, or about every one, two, three or four weeks or aboutevery one, two or three days in a therapeutic regimen. In thisembodiment, the administered dose can be the same amount of an agent, orcan be different amounts administered at several time points separatedby periods wherein the agent is not administered to the subject, orwherein a decreased amount of the agent is administered to the subject.

Regulatory T Cells (Treg): CD4+CD25+ T cells that prevent the activationand/or expansion of other cell populations, for example CD4+CD25−responder T cells. Reduction or functional alteration of Treg cellsleads to the spontaneous development of various organ-specificautoimmune diseases, including, for example, autoimmune thyroiditis,gastritis, and type 1 diabetes (see, for example, Sakaguchi et al., J.Immunol. 155:1151-64, 1995; Suri-Payer et al., J. Immunol. 160:1212-18,1998; Itoh et al., J. Immunol. 162:5317-26, 1999). The FOXP3transcription factor is predominantly expressed by the Treg cell lineage(Fontenot et al., Nature Immunol 4:330-36, 2003; Hon et al., Science299:1057-61, 2003).

Responder T Cells: A subpopulation of mature T cells that facilitate animmune response through cell activation and/or the secretion ofcytokines. In one embodiment, the responder T cells are CD4+CD25− Tcells. In another embodiment, the responder T cells are CD8+CD25− Tcells. One specific, non-limiting example of a responder T cell is a Tlymphocyte that proliferates upon stimulation by antigen or a stimulatorcell, such as an allogenic stimulator cell. Another specific,non-limiting example of a responder T cell is a T lymphocyte whoseresponsiveness to stimulation can be suppressed by Treg cells.

Sample: A portion, piece, or segment that is representative of a whole.This term encompasses any material, including for instance samplesobtained from a subject. A “biological sample” is a sample obtained froma subject. As used herein, biological samples include all clinicalsamples useful for detection of cytokine or Foxp3+ regulatory T cells insubjects, including, but not limited to, cells; tissues; bodily fluids,such as blood, derivatives and fractions of blood, such as serum; andbiopsied or surgically removed tissue, including tissues that are, forexample, unfixed, frozen, fixed in formalin and/or embedded in paraffin.In particular embodiments, the biological sample is obtained from asubject, such as blood or serum.

Subject: A human or non-human animal. In one embodiment, the subject hasan autoimmune disease, such as type 1 diabetes (T1D).

Symptom and sign: Any subjective evidence of disease or of a subject'scondition, that is, such evidence as perceived by the subject; anoticeable change in a subject's condition indicative of some bodily ormental state. A “sign” is any abnormality indicative of disease,discoverable on examination or assessment of a subject. A sign isgenerally an objective indication of disease. Signs include, but are notlimited to any measurable parameters such as tests for immunologicalstatus or the presence of lesions in a subject with an autoimmunedisease (e.g., T1D).

T Cell: A lymphoid cell that mediates cell-mediated immune responses inthe adaptive immune system. Adaptive cell-mediated immunity is immunitythat confers resistance to pathogenic conditions (including, forexample, neoplasia or infection by microbes, viruses, or bacteria) thatare not susceptible to the innate immune response (for example, notsusceptible to the antibody-making cells of the immune system). T cellsmature in the thymus, circulate between blood and lymph, populatesecondary lymphoid tissues, and are recruited to peripheral sites ofantigen exposure. T cells generally cannot recognize foreign antigenswithout the help of antigen presenting cells (APC), such as macrophages,dendritic cells or B-cells that present antigen in conjunction withmajor histocompatibility complex.

T Cell Receptor (TCR) and TCR Receptor Peptides: Membrane-bound proteinscomposed of two transmembrane chains that are found on T cells. The Tcell receptor recognizes antigen peptides presented in the context ofthe Major Histocompatibility Complex (MHC) proteins. In the case of CD4+T cells, the antigen peptides must be presented on Class II MHC, and inthe case of CD8+ T cells, the antigen peptides must be presented onClass I MHC. The T cell antigen receptor consists of either analpha/beta chain or a gamma/delta chain associated with the CD3molecular complex. The two transmembrane chains consist of two domains,called a “variable” and a “constant” domain, and a short hinge thatconnects the two domains. The V domains include V-, D-, andJ-immunoglobulin like elements in the β chain and V- and J-like elementsin the α chain.

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this can be the amount of one or more TCRpeptides useful in preventing, ameliorating, and/or treating anautoimmune disorder (e.g., MS) in a subject. Ideally, a therapeuticallyeffective amount of an agent is an amount sufficient to prevent,ameliorate, and/or treat an autoimmune disorder (e.g., MS) in a subjectwithout causing a substantial cytotoxic effect in the subject. Theeffective amount of an agent useful for preventing, ameliorating, and/ortreating an autoimmune disorder (e.g., MS) in a subject will bedependent on the subject being treated, the severity of the disorder,and the manner of administration of the therapeutic composition.

II. Overview of Aspects of the Invention

An aspect of the invention is a method for detecting in a subject apredisposition to an autoimmune disease. In one embodiment, this methodincludes detecting, in response to a peptide of Table 1, an inflammatoryresponse, with IFN-γ production, in a biological sample from the subjectthat differs from a reference level of IFN-γ production in a biologicalsample from a subject with no predisposition to an autoimmune disease.In another embodiment, this method includes detecting, in response to apeptide of Table 1, a dominant IL10 regulatory response in a biologicalsample from the subject that differs from a reference level of IL10production in a biological sample from a subject with no predispositionto an autoimmune disease. In another embodiment, this method includesdetecting, in response to a peptide of Table 1, the ratio of IFN-γ toIL10 in a biologic sample from the subject that differs from a referencelevel of IFN-γ/IL10 in a biological sample from a subject with nopredisposition to an autoimmune disease. The HLA-DQ genotype of thesubject may be determined in conjunction with detecting the response toa peptide of Table 1. In these embodiments, the HLA-DQ genotyping may beconducted prior to, after, or simultaneous with the detection of thesubject's response to a peptide of Table 1. In specific embodiments, theautoimmune disease is type 1 diabetes (T1D).

TABLE 1 Human Insulin β-chain Mimotopes SEQ Peptide ID NO SequenceDescription  1 SHLVEALYLVCGERG B:9-23 wild type human  2 SHLVEALYLVCGEEGB:9-23 mimotope; B22 arginine to glutamic acid  3 SHLVEALYLVCGGEGB:9-23 mimotope; B21-22 glutamic acid-arginine to glycine-glutamic acid 4 SHLVEELYLVCGEEG B:9-23 mimotope; B14;22 alanine to glutamic acidand arginine to glutamic acid  5 SHLVEELYLVCGERGB:9-23 mimotope; B14 alanine to glutamic acid  6 SHLVGELYLVCGERGB:9-23 mimotope; B13-14 glutamic acid-alanine to glycine-glutamic acid 7 SHLVGELYLVCGGEG B:9-23 mimotope; B13-14;21-22 glutamic acid-alanine to glycine-glutamic acid and glutamic acid-arginine to glycine-glutamic acid  8 SHLVGALYLVCGGEGB:9-23 mimotope; B13;21-22 glutamic acid toglycine and glutamic acid-arginine to glycine- glutamic acid  9SHLVGELYLVCGGRG B:9-23 mimotope; B13-14;21 glutamic acid-alanine to glycine-glutamic acid and glutamic acid to glycine 10SHLVEALYLVAGEEG B:9-23 mimotope; B19 cysteine to alanine toprevent peptide dimerization; B22 arginine to glutamic acid 11SHLVEALYLVAGGEG B:9-23 mimotope; B19 cysteine to alanine toprevent peptide dimerization; B21-22 glutamicacid-arginine to glycine-glutamic acid 12 SHLVEALYLVAGAEGB:9-23 mimotope; B19 cysteine to alanine toprevent peptide dimerization; B21-22 glutamicacid-arginine to alanine-glutamic acid 13 SHLVEALYLVAGVEGB:9-23 mimotope; B19 cysteine to alanine toprevent peptide dimerization; B21-22 glutamicacid-arginine to valine-glutamic acid 14 SHLVEALYLVAGLEGB:9-23 mimotope; B19 cysteine to alanine toprevent peptide dimerization; B21-22 glutamicacid-arginine to leucine-glutamic acid 15 SHLVEALYLVAEAEGB:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to alanine-glutamic acid- alanine-glutamic acid16 SHLVEALYLVAAEDG B:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to alanine-alanine-glutamic acid-aspartic acid 17SHLVEALYLVAQVEG B:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to alanine-glutamine-valine- glutamic acid 18SHLVEALYLVAALEG B:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to alanine-alanine-leucine- glutamic acid 19SHLVEALYLVEAEDG B:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to glutamic acid-alanine-glutamic acid-aspartic acid 20 SHLVEALYLVGQVEGB:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to glycine-glutamine-valine- glutamic acid 21SHLVEALYLVLALEG B:9-23 mimotope; B19-22 cysteine-glycine-glutamic acid-arginine to leucine-alanine-leucine- glutamic acid

Another aspect of the invention is a method for diagnosing T1D in asubject. This method includes detecting, in response to a peptide ofTable 1, an inflammatory response, with IFN-γ production, in abiological sample from the subject that differs from a reference levelof IFN-γ production in a biological sample from a control subject (i.e.,a subject known not to have T1D). In these embodiments, the detection ofIFN-γ production in response to the peptide of Table 1 is indicative ofT1D in the subject (i.e., the subject is diagnosed with T1D). In anotherembodiment, this method includes detecting, in response to a peptide ofTable 1, a dominant IL10 regulatory response in a biological sample fromthe subject that differs from a reference level of IL10 production in abiological sample from a control subject (i.e., a subject known not tohave T1D). In another embodiment, the method includes detecting, inresponse to a peptide of Table 1, an IFN-γ/IL10 ratio in a biologicalsample from the subject that differs from a reference level ofIFN-γ/IL10 in a biological sample from a control subject (i.e., asubject known not to have T1D). In these embodiments, the detection ofIFN-γ production or a ratio of IFN-γ/IL10 in response to the peptide ofTable 1 is indicative of no T1D in the subject (i.e., the subject is notdiagnosed with T1D, the subject is identified as non-diabetic). TheHLA-DQ genotype of the subject may be determined in conjunction withdetecting the response to a peptide of Table 1. In these embodiments,the HLA-DQ genotyping may be conducted prior to, after or simultaneouswith the detection of the subject's response to a peptide of Table 1.

Another aspect of the invention is a method for assessing the efficacyof a therapy for an autoimmune disease. This method includes determiningthat an inflammatory response, with IFN-γ production or IFN-γ/IL10ratio, in response to a peptide of Table 1, in a first biological sampletaken from a subject differs from the inflammatory response, with IFN-γproduction, in response to a peptide of Table 1, in a second biologicalsample taken from the subject after a period of treatment with thetherapy for the autoimmune disease, wherein a difference in theinflammatory response, with IFN-γ production or IFN-γ/IL10 in the firstbiological sample as compared to the second biological sample assessesthe efficacy of the therapy for the autoimmune disease. In a specificexample, the therapy comprises administration of a therapeuticallyeffective amount of a peptide of Table 1, including administration ofinsulin to the subject.

Another aspect of the invention is a method for inhibiting or treatingT1D. In one embodiment, the method includes administering to a subjectdiagnosed with or suspected of having or developing T1D, atherapeutically effective amount of a composition comprising a peptideof Table 1, thereby inhibiting T1D in the subject. In a specificembodiment, the composition includes a therapeutically effective amountof a peptide of SEQ ID NO:2. In related embodiments, the subject isfirst identified as having T1D by methods that include detecting, inresponse to a peptide of Table 1, an inflammatory response, with IFN-γproduction or an IFN-γ/IL10 ratio, in a biological sample from thesubject.

Another aspect of the invention is a method for inducingimmunosuppression in a subject having or suspected of having anautoimmune disease. This method includes administering to a subject atherapeutically effective amount of a composition comprising a peptideof Table 1, thereby inducing immunosuppression in the subject. Inspecific embodiments, the subject has, or is suspected of developing,T1D. In specific embodiments, the autoimmune disease is T1D. In relatedembodiments, the subject is first identified as having T1D by methodsthat include detecting, in response to a peptide of Table 1, aninflammatory response, with IFN-γ production or IFN-γ/IL10 ratio, in abiological sample from the subject.

Diagnostic Methods and Method for Monitoring Treatment

It is disclosed herein that T cell responses to contact with a peptideof Table 1 differ between subjects with type 1 diabetes (T1D) andhealthy controls (i.e., non-diabetic subjects). Accordingly, it is nowpossible to use these T cell responses to detect T1D, or a predilectionto T1D in a subject, and/or to monitor the efficacy of T1D therapies.These methods can include determining whether the T cell responses inone or more biological samples taken from a subject differ from eachother or from another reference point. The reference point can be astandard value, or a control with a known T cell response (i.e., a Tcell response from a subject confirmed type diabetic or non-diabetic).However, the reference point can also be another sample from the samesubject. For example, prior to the onset of a T1D therapy, a firstsample is taken from the subject. Following onset of therapy, a secondsample is taken from the subject. The T cell responses are evaluated inthe first sample and in the second sample. If the T cell responses areindicative of an inflammatory response (e.g., with IFN-γ production orincreased IFN-γ/IL10 ratio) in the second sample as compared to thefirst sample, the therapy is not having the desired effect (and thuscould be discontinued). However, if the T cell responses are indicativeof a reduction in inflammatory response (e.g., with reduced IFN-γproduction or IFN-γ/IL10) or an IL10 regulatory T cell response in thefirst sample as compared to the second sample, then the therapy ishaving the desired effect (and thus may be continued).

A biological sample that is useful in these methods includes any part ofthe subject's body that can be obtained and reduced to a form that canbe analyzed for the T cell response. Typically, a biological sample willcontain T cells in amounts sufficient to conduct the desired analysis.The T cells of use in these methods can be derived from any convenient Tcell source in the subject, such as lymphatic tissue, spleen cells,blood, or pancreas. The T cells can be enriched, if desired, by standardpositive and negative selection methods. If enriched, the T cellpopulation should retain a sufficient number of antigen-presenting cellsto present the TCR peptide to the regulatory T cells. A convenientsource of T cells to use in these methods are peripheral bloodmononuclear cells (PBMC), which can be readily prepared from blood bydensity gradient separation, by leukapheresis or by other standardprocedures known in the art. Thus, suitable biological samples include,for example, blood, or the components of blood, such as serum orisolated white blood cells. The biological sample may contain T cells inthe peripheral blood. In specific embodiments, the biological sample isunfractionated peripheral blood mononuclear cells (PBMCs). Biologicalsamples can be obtained from normal, healthy subjects or from subjectswho are predisposed to or who are suffering from T1D. The disclosedmethods contemplate as a subject any living organism capable ofexperiencing an autoimmune disease, including veterinary subjects (suchas, felines, canines, rodents (e.g., mice and rats), equines, bovines,ovines, and the like) and human subjects (including, adults,adolescents, and children).

In one embodiment, at least two biological samples are obtained from asingle subject over time, such as during a therapeutic regimen. In onenon-limiting example, the samples are obtained from the same subjectduring the administration of a pulsatile doses of any therapeutic agent.The T cell response to a peptide of Table 1 is assessed in the firstsample and the second sample. An absent or reduced T cell responseindicative of inflammation in the second sample as compared to the firstsample indicates that the therapy is effective. An increased orsubstantially similar T cell response indicative of inflammation in thesecond sample indicates that the therapy is ineffective.

A variety of T1D therapies that are administered over a specified timeperiod can be evaluated using the methods disclosed herein. In someembodiments, at least two biological samples are obtained from a singlesubject over time, such as during a therapeutic regimen. In oneembodiment, the samples are obtained from the same subject during theadministration of a maintenance therapy. A reduction in the inflammatoryT cell response to contact with a peptide of Table 1 in the secondsample as compared to the first sample indicates that the therapy iseffective, and maintains desired clinical effect. A substantial decreaseor elimination of the T cell response indicates that the therapeuticagent is effective and suggests that the dose of the therapeutic agentcould be lowered to possibly achieve the desired therapeutic effect. Anincrease in the inflammatory T cell response to contact with a peptideof Table 1 in the second sample as compared to the first sampleindicates that the therapy is not effective, and indicates that the doseof the agent is insufficient or that a different therapeutic agentshould be utilized in the subject.

The T cell response to contact with a peptide of Table 1 can be detectedin a variety of methods known to those of skill in the art for thedetection of T cell responses indicative of inflammation, including theexpression of specific cytokines.

In particular examples, the level, ratio, or activity of IFN-γ and/orIL10 production is detected in response to contact with the nativeinsulin B:9-23 peptide and one or more insulin mimotopes set forth inTable 1. IFN-γ and/or IL10 protein(s) may be evaluated by standardmethods (for example, using an antibody array, immunofluorescence,Western blot, radioimmunoassay, sandwich immunoassays (including ELISA),Western blot, affinity chromatography (affinity ligand bound to a solidphase), in situ detection with labeled antibodies, or any of a number offunctional assays described herein).

An inflammatory T cell response (measured by evaluation of a nucleicacid transcript or protein level) and/or activity (protein) may bedifferent with respect to a reference level of expression and/oractivity of a specific cytokine. A variety of reference points can beused. In some instances, a reference point is the expression and/oractivity of the cytokine in a biological sample collected from a subjectnot suffering from an autoimmune disease (such as a control subject). Inother examples, a reference point is an average (or “normal-range”)value for the expression and/or activity of the cytokine in subjects notsuffering from an autoimmune disease, which normal-range value has beendetermined from population studies. The control may be a standard value,such as a sample with a known amount of mRNA or protein. In particularapplications, such as some methods for determining the efficacy of anautoimmune disease therapy, a reference also can be, for example, theexpression and/or activity of a cytokine in a biological sample from thesubject prior to onset of the therapy, and/or after some period of timefollowing (or during) the therapy. Alternatively, the efficacy of anautoimmune disease therapy can be determined by comparing the expressionand/or activity of the cytokine in a test subject, who is receivingtherapy, as compared to a second subject suffering from an autoimmunedisease, who is receiving a placebo rather than therapy. In this lattersituation, it is expected that the expression levels and/or activitiesof the cytokine in the treated subject would diverge from those of aplacebo-treated subject, with such expression levels and/or activitiesin an effectively treated subject approaching corresponding valuesobserved in a healthy control subject. The autoimmune disease may beT1D. The cytokine may be at least one of IFN-γ and/or IL10.

The expression level and/or activity of the cytokine (e.g., gene,transcript or protein) may differ from a reference expression leveland/or activity by at least ±10%; for example, by at least about ±15%,at least about 25%, at least about 40%, at least about ±50%, at leastabout ±60%, at least about ±75%, or at least about ±90%.

In methods of this disclosure, IFN-γ levels are measured. A variety ofmethods can be used to detect and quantify IFN-γ expression by T cells.In some embodiments, IFN-γ mRNA is measured. IFN-γ mRNA can be measuredby any method known to one of skill in the art. For example, polymerasechain reaction (PCR) can be used. Briefly, total RNA is extracted from Tcells by any one of a variety of methods well known to those of ordinaryskill in the art. Sambrook et al. (In Molecular Cloning: A LaboratoryManual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocolsin Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992)provide descriptions of methods for RNA isolation. The extracted RNA isthen used as a template for performing reverse RT-PCR amplification ofIFN-γ cDNA. IFN-γ-specific primers for the PCR reaction can be obtained,for example, from Applied Biosystems (Foster City, Calif.). Methods andconditions for PCR are described in Kawasaki et al., (In PCR Protocols,A Guide to Methods and Applications, Innis et al. (eds.), 21-27,Academic Press, Inc., San Diego, Calif., 1990). In other examples,Northern blotting or RNA dot blots can also be used to detect IFN-γmRNA.

Additional methods for measuring IFN-γ expression levels utilizesmeasurements of IFN-γ protein. Antibodies to IFN-γ can be used inmethods such as immunoassays (for example RIAs and ELISAs),immunohistochemistry, and Western blotting to assess the expression ofIFN-γ.

For Western blotting, total cellular protein is extracted from T cellsand electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. Theproteins are then transferred to a membrane (for example, nitrocelluloseor PVDF) by Western blotting, and an anti-IFN-γ antibody (e.g., a rabbitanti-human IFN-γ antibody) preparation is incubated with the membrane.After washing the membrane to remove non-specifically bound antibodies,the presence of specifically bound antibodies is detected by the use of(by way of example) an anti-rabbit antibody conjugated to an enzyme suchas alkaline phosphatase. Application of an alkaline phosphatasesubstrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazoliumresults in the production of a dense blue compound by immunolocalizedalkaline phosphatase.

One method embodiment for detecting or diagnosing in a subject anautoimmune disease, or a predisposition to an autoimmune disease,involves (a) determining the expression and/or activity of IFN-γ (e.g.,gene, transcript and/or protein) in a biological sample from a subject;and (b) comparing the expression and/or activity of the IFN-γ in thebiological sample to the expression and/or activity of the IFN-γ in areference sample, wherein a difference in the expression and/or activityof the IFN-γ in the biological sample and the reference sample detectsor diagnoses an autoimmune disease or a predisposition to an autoimmunedisease in the subject.

In another method embodiment, the efficacy of an autoimmune diseasetherapy can be determined by (a) obtaining a first biological samplefrom a first subject suffering from an autoimmune disease; (b) treatingthe first subject with a candidate therapy; (c) obtaining a secondbiological sample from at least one of the following: (i) the firstsubject following treatment; (ii) an individual not suffering from anautoimmune disease; or (iii) a second subject suffering from anautoimmune disease receiving a placebo rather than therapy; and (d)comparing the expression and/or activity of IFN-γ in the first andsecond biological samples after contact with a peptide of Table 1,wherein a change in the expression and/or activity of IFN-γ indicatesthat the candidate therapy is effective at treating the autoimmunedisease in the first subject. In other methods, steps (a)-(d) can berepeated on the first subject after altering the dose or dosing regimenof the candidate therapy.

In more specific embodiments, a method for monitoring an outcome of anautoimmune disease therapy in a subject, involves (a) obtaining a firstbiological sample from a subject suffering from T1D; (b) treating thesubject with a T1D therapy; (c) obtaining a second biological samplefrom the subject following a period of treatment with the T1D therapy;and (d) comparing the expression and/or activity of IFN-γ in response tocontact with a peptide of Table 1, in the first and second biologicalsamples, wherein a relative change in the expression and/or activity ofIFN-γ in the first and second biological sample monitors an outcome ofthe candidate therapy.

In some embodiments, T cells are isolated from the sample prior toperforming the assay for T cell response to contact with a peptide ofTable 1. The T cells can be any T cells of interest, such as, but notlimited to, CD3+, CD4+, and/or CD25+ T cells. In one specificnon-limiting example, CD4+CD25+ T cells can be isolated, and theexpression of IFN-γ can be assessed in the CD4+CD25+ T cells.

Methods for the isolation and quantitation of T cells, are well known inthe art. Typically, labeled antibodies specifically directed to one ormore cell surface markers are used to identify and quantify the T cellpopulation. The antibodies can be conjugated to other compoundsincluding, but not limited to, enzymes, magnetic beads, colloidalmagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs. The enzymes that can be conjugated to the antibodiesinclude, but are not limited to, alkaline phosphatase, peroxidase,urease and β-galactosidase. The fluorochromes that can be conjugated tothe antibodies include, but are not limited to, fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate,phycoerythrin (PE), allophycocyanins and Texas Red. For additionalfluorochromes that can be conjugated to antibodies see Haugland, R. P.,Handbook of Fluorescent Probes and Research Products, published byMolecular Probes, 9^(th) Edition (2002). The metal compounds that can beconjugated to the antibodies include, but are not limited to, ferritin,colloidal gold, and particularly, colloidal superparamagnetic beads. Thehaptens that can be conjugated to the antibodies include, but are notlimited to, biotin, digoxigenin, oxazalone, and nitrophenol. Theradioactive compounds that can be conjugated or incorporated into theantibodies are known to the art, and include, but are not limited to,technetium 99 (⁹⁹Tc), ¹²⁵I, and amino acids comprising anyradionuclides, including, but not limited to, ¹⁴C, ³H and ³⁵S.

Fluorescence activated cell sorting (FACS) can be used to sort cellsthat are CD4+, CD25+, or both CD4+ and CD25+, by contacting the cellswith an appropriately labeled antibody. However, other techniques ofdiffering efficacy may be employed to purify and isolate desiredpopulations of cells. The separation techniques employed should maximizethe retention of viability of the fraction of the cells to be collected.The particular technique employed will, of course, depend upon theefficiency of separation, cytotoxicity of the method, the ease and speedof separation, and what equipment and/or technical skill is required.

Additional separation procedures may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and “panning,” which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique. Antibodiesattached to magnetic beads and other solid matrices, such as agarosebeads, polystyrene beads, hollow fiber membranes and plastic Petridishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell known in the art.

Unbound cells then can be eluted or washed away with physiologic bufferafter sufficient time has been allowed for the cells expressing a markerof interest (e.g., CD4 and/or CD25) to bind to the solid-phase linkedantibodies. The bound cells are then separated from the solid phase byany appropriate method, depending mainly upon the nature of the solidphase and the antibody employed, and quantified using methods well knownin the art. In one specific, non-limiting example, bound cells separatedfrom the solid phase are quantified by FACS.

Antibodies may be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with FACS to enable cell separation and quantitation, as knownin the art.

In additional embodiments, cytokine expression levels in the biologicalsample of interest are measured using any one of a variety of standardmethods used to detect and quantify cytokine expression by T-cells. Forexample, an immunospot assay, such as the enzyme-linked immunospot or“ELISPOT” assay, can be used. The immunospot assay is a highly sensitiveand quantitative assay for detecting cytokine secretion at the singlecell level. Immunospot methods and applications are well known in theart and are described, for example, in Czerkinsky et al., J. Immunol.Methods 110:29-36, 1988; Olsson et al. J. Clin. Invest. 86:981-985,1990; and EP 957359.

The immunospot assay uses microtiter plates containing membranes thatare precoated with a capture agent, such as an anti-cytokine antibody,specific for the cytokine to be detected. T cells of interest are platedtogether with a composition (e.g., an effective amount of a peptide ofTable 1). The T cells that respond to the composition secrete variouscytokines. As a cytokine to be quantified is locally released by the Tcells, it is captured by the membrane-bound antibody. After a suitableperiod of time the cell culture is terminated, the T cells are removedand the plate-bound cytokine is visualized by an appropriate detectionsystem. Each cytokine-secreting T cell will ideally be represented as adetectable spot. The number of spots, and thus the number of T cellssecreting the particular cytokine of interest, can be counted manually(for example, by visualization via light microscopy) or by using anautomated scanning system (for example, an Immunospot Reader).

Variations of the standard immunospot assay are well known in the artand can be used to detect alterations in cytokine production in themethods of the disclosure. For example, U.S. Pat. No. 5,939,281 (whichis incorporated herein by reference) describes an improved immunospotassay that uses a hydrophobic membrane instead of the conventionalnitrocellulose membrane, to bind the cytokine capture reagent. Thisvariation can be used to reduce the non-specific background and increasethe sensitivity of the assay. Other modifications to the standardimmunospot assay that increase the speed of processing multiple samples,decrease the amount of reagents and T cells needed in the assay, orincrease the sensitivity or reliability of the assay, are contemplatedherein and can be determined by those skilled in the art.

U.S. Pat. No. 6,218,132 (which is incorporated herein by reference)describes a modified immunospot assay in which T cells are allowed toproliferate in response to stimulation before detection of the cytokineof interest. This method, although more time-consuming, can be used toincrease the sensitivity of the assay for detecting T cells present at alow frequency in the starting population.

Antibodies suitable for use in immunospot assays, which are specific forsecreted cytokines (such as IFN-γ and/or IL10), as well as detectionreagents and automated detection systems, are well known in the art andgenerally are commercially available. Appropriate detection reagents arealso well known in the art and commercially available, and include, forexample, secondary antibodies conjugated to fluorochromes, coloredbeads, and enzymes whose substrates can be converted to colored products(for example, horseradish peroxidase and alkaline phosphatase). Othersuitable detection reagents include secondary agents conjugated toligands (for example, biotin) that can be detected with a tertiaryreagent (for example, streptavidin) that is detectably labeled as above.

Other methods for detecting and quantifying cytokine expression are wellknown in the art, and can be used as an alternative to immunospotassays. Such methods include the enzyme-linked immunoabsorbent assay(ELISA), which can be used to measure the amount of cytokine secreted byT cells into a supernatant (see, e.g., Vandenbark et al., Nature Med.2:1109-1115, 1996). Alternatively, the expression of cytokine mRNA canbe determined by standard immunological methods, which include reversetranscriptase polymerase chain reaction (RT-PCR) and in-situhybridization (as described above).

In the methods disclosed herein, suppression of cell proliferation by Tcells from the sample of interest can also be measured. Suppression ofproliferation can be evaluated using many methods well known in the art.In one embodiment, T cell proliferation is quantified by measuring[³H]-thymidine incorporation. Proliferating cells incorporate thelabeled DNA precursor into newly synthesized DNA, such that the amountof incorporation, measured by liquid scintillation counting, is arelative measure of cellular proliferation. In another embodiment, cellproliferation is quantified using the thymidine analogue5-bromo-2′-deoxyuridine (BrdU) in a proliferation assay. BrdU isincorporated into cellular DNA in a manner similar to thymidine, and isquantified using anti-BrdU mAbs in an ELISA.

Method for Inhibiting an Autoimmune Disease

Another aspect of the invention provides methods for inhibiting anautoimmune disease. These methods include administering to a subject inneed thereof a therapeutically effective amount of a composition thatdecreases T-cell inflammatory response to contact with a peptide ofTable 1, thereby inhibiting the autoimmune disease. The autoimmunedisease may be T1D.

The composition can include a single peptide of Table 1, or multiplepeptides of Table 1. The peptides may include peptides having at least90% homology to a peptide sequence of those peptides set forth in Table1, and which stimulate T cells that are reactive against insulin, toconvert naive T cells into Foxp3+ regulatory T cells and preventdiabetes onset. The peptides may include fragments of the peptides ofTable 1 that retain the ability to stimulate T cells that are reactiveagainst insulin, to convert naive T cells into Foxp3+ regulatory T cellsand prevent diabetes onset. The peptides may include insulin peptides,including insulin, proinsulin, and/or the insulin β chain, having atleast one amino acid substitution of SEQ ID NOs: 2-9, and whichstimulate T cells that are reactive against insulin, to convert naive Tcells into Foxp3+ regulatory T cells and prevent diabetes onset. Anexemplary peptide includes the amino acid sequence set forth in SEQ IDNO:2. Appropriate peptides to use in the methods disclosed herein can bedetermined by those skilled in the art. The immunogenicity of a givenpeptide can be predicted using well-known algorithms that predict T cellepitopes (see, e.g., Savoie et al., Pac. Symp. Biocomput. 1999:182-89,1999; Cochlovius et al., J. Immunol. 165:4731-41, 2000). Both theimmunogenicity and the specificity of a given peptide can be confirmedby standard immunological assays that measure in vivo or in vitro T cellresponses (e.g., T cell proliferation assays, delayed typehypersensitivity assays, ELISA assays, ELISPOT assays and the like).

The composition(s) containing the peptide(s) can be formulated as avaccine. In these embodiments, the formulation may also include atherapeutically effective amount of an adjuvant, such as, but notlimited to, complete Freund's adjuvant (CFA), incomplete Freund'sadjuvant (IFA), immunomodulatory oligonucleotides including Immunomers(Wang et al., Int J Oncol 2004, 24: 901-08.) and CpGoligodeoxynucleotides (Mosemann et al., J. Immunol. 173:4433, 2004), orIVX-908 (ID Biomedical of Canada). Further additional agents that can beadministered to the subject include, for example, a therapeuticallyeffective amount of: an interferon (such as IFN β1a or IFN β1b), aninterleukin (such as IL-4), an antibody to an interleukin (such asanti-IL-12 or anti-IL-23), Glatiramer acetate (also known as Copolymer1), Natalizumab, and/or Mitoxantrone.

In one embodiment, an additional therapeutic agent is administered tothe subject with an autoimmune disorder. These therapeutic agents can beadministered at the same time, or at a different time (sequentially) asthe peptide of Table 1 that increases the production of Foxp3+regulatory T cells. These agents may include, but are not limited to,interferon-beta. These agents can be included in the same composition asthe peptide of Table 1 that increases the production of Foxp3+regulatory T cells, or can be administered in separate compositions.

Administration of a therapeutically effective amount of the peptide ofTable 1 that increases the production of Foxp3+ regulatory T cells canbe utilized whenever desired, for example, at the first sign of symptomsof an autoimmune disease, such as T1D, or at the first sign of symptomsof inflammation or insulin-specific antibodies, or T cell mediateddestruction of insulin producing beta cells within pancreatic islets, orthe detection of CD4 T cells targeting pancreatic beta cells, or thepresence of CD4 T cells in the pancreas and/or pancreatic lymph nodes ofa subject.

Alternatively, administration of a therapeutically effective amount ofthe peptide of Table 1 that increases the production of Foxp3+ can bedone prophylactically (i.e., before any overt systems of autoimmunedisease onset).

Therapeutically effective amounts of the peptide of Table 1 thatincreases the production of Foxp3+ can be administered by a number ofroutes, including parenteral administration, for example, intravenous,intraperitoneal, intramuscular, intradermal, intrasternal, orintraarticular injection, or infusion. One of skill in the art canreadily determine the appropriate route of administration.

The therapeutically effective amount of the peptide of Table 1 thatincreases the production of Foxp3+ will be dependent upon the subjectbeing treated, the severity and type of the affliction, and the mannerof administration. For example, a therapeutically effective amount of apeptide of Table 1 can vary from about 1-500 μg/injection. The exactamount of the peptide is readily determined by one of skill in the artbased on the age, weight, sex, and physiological condition of thesubject. Effective doses can be extrapolated from dose-response curvesderived from in vitro or animal model test systems.

Generally, a therapeutically effective amount of the peptide of Table 1that increases the production of Foxp3+, is that amount of the peptidethat inhibits the advancement, or causes regression of T1D, or which iscapable of relieving symptoms caused by an autoimmune disease. Forexample, a therapeutically effective amount of the peptide of Table 1that increases the production of Foxp3+, is that amount of the peptidethat is capable of inducing tolerance in a diabetic or pre-diabeticsubject.

The peptide of Table 1 that increases the production of Foxp3+ can beadministered in a pharmaceutically acceptable carrier, such as bufferedsaline or another medium suitable for administration to a subject. Forexample, one or more peptides of Table 1 can be administered in apharmaceutically acceptable carrier, such as a carrier formulated forinjection. It should be noted that a single peptide of Table 1 thatincreases the production of Foxp3+ can be administered, or multiplepeptides can be administered to a subject of interest (such as a subjectwith or suspected of having T1D).

In one embodiment, the peptide of Table 1 that increases the productionof Foxp3+ can be administered in conjunction with one or more additionalpharmaceutical agents. The additional pharmaceutical agents can beadministered at the same time as, or sequentially with, the peptide ofTable 1. In one embodiment, the additional pharmaceutical agent is anadditional immunosuppressive agent. When administered at the same time,the additional pharmaceutical agent(s) can be formulated in the samecomposition that includes the peptide of Table 1. For, example,additional pharmaceutical agents may include immunosuppressive agents(for example, azathioprine or glucocorticoids, such as dexamethasone orprednisone), anti-inflammatory agents (for example, glucocorticoids suchas hydrocortisone, dexamethasone or prednisone, or non-steroidalanti-inflammatory agents such as acetylsalicylic acid, ibuprofen ornaproxen sodium), cytokines (for example, interleukin-10 andtransforming growth factor-β), or a vaccine.

Those skilled in the art can determine an appropriate time and durationof therapy that includes the administration of a peptide of Table 1 toachieve the desired preventative or ameliorative effects on the immunepathology.

In a specific embodiment, the method includes administering to a subjecta therapeutically effective amount of a peptide comprising SEQ ID NO:2to induce immunosuppression in a subject. An adjuvant can optionally beincluded with the peptide comprising SEQ ID NO:2.

Immunosuppression in the subject can be evaluated using methods wellknown in the art. In one embodiment, a white blood cell count (WBC) isused to determine the responsiveness of the subject's immune system. AWBC measures the number of white blood cells in a subject. Using methodswell known in the art, the white blood cells in a subject's blood sampleare separated from other blood cells and counted. Normal values of whiteblood cells are about 4,500 to about 10,000 white blood cells/μl. Lowernumbers of white blood cells can be indicative of a state ofimmunosuppression in the subject.

In another embodiment, immunosuppression in a subject may be determinedusing a T lymphocyte count. Using methods known in the art, the whiteblood cells in a subject's blood sample are separated from other bloodcells. T lymphocytes are differentiated from other white blood cellsusing standard methods in the art, such as, for example,immunofluorescence or FACS. Reduced numbers of T cells, or a specificpopulation of T cells can be used as a measurement of immunosuppression.In one embodiment the population of T cells monitored or suppressed arethose T cells that recognize and destroy insulin producing beta cells inpancreatic islets. A reduction in the number of such T-cells, or in thegeneral population of T cells, compared to the number of T cells (or thenumber of cells in the specific population) prior to treatment can beused to indicate that immunosuppression has been induced.

In another embodiment, effective treatment or inhibition of inflammationor immunosuppression in the subject can be assayed by measuring cytokinelevels in the subject. Cytokine levels in body fluids or cell samplesare determined by conventional methods. For example, an immunospotassay, such as the enzyme-linked immunospot or “ELISPOT” assay, asdescribed herein, can be used. The cytokine(s) measured may include atleast one of IFN-γ and IL10.

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for the purposes of illustration of aspects of the embodiments ofthe present invention. The examples are not intended to limit theinvention, as one of skill in the art would recognize from the aboveteachings and the following examples that other techniques and methodscan satisfy the claims and can be employed without departing from thescope of the claimed invention.

EXAMPLES Example 1: Detection of Robust IFN-v Responses to an InsulinMimotope

Subjects with new-onset T1D (n=28) were recruited from the Barbara DavisCenter for Diabetes clinics. Non-diabetic controls (n=27) were healthyadult volunteers negative for all islet autoantibodies. The studyprotocol was approved by the Institutional Review Board and writteninformed consent was obtained from all study participants. The T1Dsubjects had a very short duration of diabetes with the mean time fromdiagnosis only 15 days; 26/28 (93%) T1D individuals had diabetes lessthan 3 weeks prior to assays being performed. All subjects were HLAgenotyped. Islet autoantibodies to insulin, GAD65, IA-2, and ZnT8 weremeasured from the serum by radioimmunoassay as previously described(42). HLA-DRB, DQA, and DQB genotyping was performed using linear arraysof immobilized sequence-specific oligonucleotides similar to previouslydescribed methodology (43). Demographic and clinical characteristics aresummarized in the following table:

TABLE 2 Clinical characteristics, islet autoantibody status, and HLAgenotype of study participants Type 1 Diabetes No DiabetesCharacteristic (n = 28) (n = 27) P-value Age, years Mean (SD) 16.1 (7.3)27.7 (11.2) <0.01  Median 14 23 Range 10-49 14-53 Gender, Number (%)Male 19 (68) 14 (52) 0.28 Female 9 (32) 13 (48) Diabetes Duration, daysMean (SD) 15 (22.1) NA NA Median 11 Range  0-114 Islet AutoantibodyPositive, Number (%) 25 (89.3) 0 (0) NA HLA-DQ Genotype, Number (%) Twoalleles with non- 17 (61) 9 (33) 0.11 β57Asp^(A) One allele with non- 9(32) 14 (52) β57Asp No alleles with non- 2 (7) 4 (15) β57Asp^(A)DQβ*03:02, 02:01, 02:02, 05:01, and 06:04 lack β57 aspartic acid.

The non-diabetic control subjects are slightly older than the T1Dpatients, and contain more individuals having ‘diabetogenic’ DQ alleles,which have a polymorphism at position 57 in the β chain (non-β57Asp),than expected in the general population to allow for comparisons betweenT1D and control subjects. FIG. 1A outlines the study participants andtheir given DQ genotypes.

Cytokine enzyme-linked immunosorbent spot (ELISPOT) responses fromunfractionated peripheral blood mononuclear cells (PBMCs) were measuredas previously described using the human IFN-γ and IL10 ELISPOT kits(UCyTech Biosciences) (44). Briefly, freshly isolated PBMCs (1×106) werecultured in 250 μl of serum free AIM-V® Medium (Invitrogen) and 10 μM ofpeptide which were dissolved in PBS. The cells were supplemented with anadditional 250 μl of medium after 24 h, and harvested 24 h later. Afterwashing, the cells were resuspended in 300 μl medium and transferred asthree 100 μl aliquots to 96-well clear polystyrene culture plates coatedwith the appropriate cytokine capture monoclonal antibody andsubsequently treated with 1×blocking solution (UCyTech). Seventeen hourslater, the cells were removed by decanting, and the wells washed (2×PBS,and 5×PBS containing 0.05% Tween-20). Spots were then formed bysequential incubations with the biotinylated 2nd site anti-IFN-γ oranti-IL10, gold-labeled goat anti-biotin, and a precipitating silversubstrate. Spots were enumerated with a Bioreader 4000 Pro X (BIOSYSGmbH). No antigen wells were a negative control and 1 μl of the Pentacelvaccine (Sanofi Pasteur) was used as a positive control stimulus in eachassay.

Total spot numbers from ELISPOT assays were analyzed with anonparametric Mann-Whitney test (rank sum test). ELISPOT response ratesbetween T1D and controls for a given condition were compared with atwo-sided Fisher's exact text. For CFSE proliferation assays, a Wilcoxonsigned rank test compared samples from the same subject. For allstatistical tests, a two-tailed p value of <0.05 is consideredsignificant. Analyses were performed using GraphPad Prism 4.0 software.

We measured IFN-γ and IL10 ELISPOT responses to the native insulinB:9-23 peptide and two insulin mimotopes (Tables S1 and S2).

TABLE S1 IFN-γ and IL10 ELISPOT responses to insulin peptides by HLAgenotype in new onset type 1 diabetes subjects Patient Data HLA DR andDQ Alleles Age T1D Allele1 Allele2 IFN-γ Total ELISPOTS Case (yrs) Sex(Days) DRB1 DQA1 DQB1 DRB1 DQA2 DQB2 No Ag Pentcel  1 12 M 8 404 301 302301 501 201 3 350  2 13 M 114 401 301 302 101 101 501 2 223  3 14 M 12401 301 302 101 101 501 2 181  4 20 M 15 301 501 201 301 501 201 13 284 5* 14 M 9 301 501 201 101 101 501 2 113  6 16 M 12 401 301 302 801 301302 0 181  7 19 M 6 401 301 302 701 201 202 3 160  8 14 M 14 401 301 302701 201 202 1 278  9 14 M 11 301 501 201 301 501 201 0 138 10 14 M 60301 501 201 1302 102 604 1 87 11 12 F 7 301 501 201 101 101 501 5 168 1216 F 6 401 301 302 301 501 201 5 370 13 13 F 0 401 301 302 301 501 201 8300 14 21 F 8 301 501 201 1302 102 604 0 206 15 15 F 1 301 501 201 1302102 604 6 357 16 15 M 14 404 301 302 407 301 302 0 440 17 14 M 9 301 501201 301 501 201 1 305 18 27 M 0 404 301 302 1301 103 602 4 283 19* 11 F18 407 301 302 1406 501 301 4 270 20 16 M 11 301 501 201 404 301 301 041 21 49 M 15 401 301 302 801 401 402 3 458 22 16 F 14 701 201 202 401301 301 2 152 23 14 M 7 405 301 302 801 401 402 2 96 24 14 M 14 101 101501 1304 501 301 0 68 25 12 M 12 401 301 302 302 401 402 1 300 26 10 F 0101 101 501 401 301 301 0 413 27* 15 M 12 401 301 301 1104 501 301 0 12828 12 F 7 1602 102 502 1102 501 301 0 187 IFN-γ Total ELISPOTS IL-10Total ELISPOTS B: 9-23 B: 9-23 B22E B22E Case Wt B22E 21G No Ag PentacelWt B22E 21G  1 5 26 4 — — — — —  2 3 143 6 — — — — —  3 5 70 6 — — — — — 4 35 151 34 — — — — —  5* 1 2 0 — — — — —  6 1 4 1 — — — — —  7 7 6 4 —— — — —  8 0 4 2 — — — — —  9 1 30 1 — — — — — 10 1 6 1 — — — — — 11 638 2 — — — — — 12 13 15 7 — — — — — 13 11 6 4 3 160 1 1 1 14 154 3 1 1301 1 70 2 15 0 6 1 0 545 2 6 1 16 4 12 0 1 304 0 161 3 17 1 3 0 1 303 07 4 18 5 163 2 — — — — — 19* 4 30 11 — — — — — 20 1 4 0 — — — — — 21 619 5 — — — — — 22 1 8 0 — — — — — 23 1 19 10 — — — — — 24 0 4 1 0  88 623 3 25 0 2 1 0 521 1 7 1 26 1 10 2 2 350 0 1 4 27* 3 27 5 — — — — — 284 27 8 — — — — — *Denotes islet autoantibody negative subject Underlinedand bold allele contains non-β57Asp

TABLE S2 IFN-γ and IL10 ELISPOT responses to insulin peptides by HLAgenotype in control subjects without diabetes Patient HLA DR and DQAlleles IFNg Total ELISPOTS IL-10 Total ELISPOTS Data B: 9-23 B: 9-23Age Allele1 Allele2 No Pen- B22E No Pen- B22E Case (yrs) Sex DRB1 DQA1DQB1 DRB1 DQA2 DQB2 Ag tcel Wt B22E 21G Ag tacel Wt B22E 21G  1 21 M 404301 302 101 101 501 4 136 1 34 8 1 88 3 31 15  2 18 F 401 301 201 701201 202 4 154 4 2 32 High Background  3 15 M 404 301 302 701 201 202 1135 0 0 1 11  30 6 7 0  4 14 F 404 301 302 301 501 201 0 227 3 1 26 2 354 5 23  5 32 F 402 301 302 301 501 201 0 302 1 2 1 4 305 3 13 5  6 24 F301 501 201 901 301 201 5 300 4 3 5 4 311 120 66 63  7 29 F 102 101 5011201 101 501 3 307 8 10 5 3 304 3 11 4  8* 23 F 301 501 201 701 201 2021 304 0 0 0 2 311 0 11 4  9 22 M 301 501 201 101 101 501 1 358 0 9 0 3330 30 13 5 10 17 M 101 101 501 408 301 301 0 86 2 123 10 4 93 4 115 2711 45 F 404 301 302 1101 501 301 3 111 5 86 11 5 27 4 221 43 12 53 F 401301 302 404 301 301 0 44 0 2 6 2 16 6 44 3 13 41 M 403 301 302 1501 102601 2 98 4 12 3 3 49 8 27 16 14* 50 F 1302 102 604 401 301 301 0 13 0 00 3 20 7 24 10 15* 53 M 404 301 302 1501 102 602 0 17 1 0 3 2 15 12 5211 16 33 F 1302 102 604 801 401 402 0 211 0 2 0 7 164 0 5 1 17 24 F 101101 501 401 301 301 1 465 0 16 2 4 321 22 17 11 18 25 M 103 101 501 1501102 602 0 303 2 39 0 0 281 1 43 0 19* 16 M 402 301 202 1104 501 301 1314 7 73 5 0 162 7 31 6 20 23 M 701 201 202 901 301 303 0 406 2 89 2 3322 4 113 7 21 23 M 301 501 201 1501 102 602 1 326 0 19 4 3 357 1 231 1122 22 F 101 101 501 1502 103 601 0 313 0 7 2 1 307 3 108 9 23 23 M 102101 501 1501 102 602 0 301 2 25 1 2 303 3 31 5 24 30 M 401 301 301 1103501 301 0 304 0 6 0 1 310 11 80 20 25 23 F 1101 501 301 1101 501 301 0300 0 9 1 3 348 7 13 8 26 26 M 1501 102 602 1301 103 603 2 304 1 175 1 1301 1 319 3 27 23 M 1104 501 301 1501 102 602 0 314 0 10 0 4 312 1 29 6*Denotes a first degree relative with T1D Underlined and bold allelecontains non-β57Asp

The native amino acid sequence of insulin B chain amino acids 9-23 islisted in FIG. 1B along with two mimotopes designed to bind‘diabetogenic’ DQ alleles, mainly DQ8 and DQ2, in an unfavorable bindingposition or ‘register.’ A peptide can bind in multiple positions orregisters with amino acids occupying positions p1-p9 in the peptidebinding groove (18, 19). A peptide is anchored by amino acids binding todistinct structural pockets at position 1, 4, 6, and 9 of the MHC classII peptide binding groove, while the remaining amino acids can interactwith a T cell receptor (20, 21). It is well established that subtlestructural changes to pocket 9 in the peptide binding groove influenceT1D susceptibility in mice and humans. The two mimotopes studied areinsulin B:9-23 (B22E) and B:9-23 (B21G, 22E) as the amino acidsubstitutions allow the mimotopes to be anchored at pocket 9, as B22arginine of the native peptide is an otherwise unfavorable match (FIG.1B).

Cytokine ELISPOT results were obtained from all T1D and control subjectsbased upon DQ genotype with alleles containing β57 aspartic acid.Freshly isolated PBMCs were cultured in the presence or absence of asingle insulin peptide for 48 hours, washed, and then cells transferredto an IFN-γ or IL10 monoclonal antibody coated plate for overnightculture followed by development and enumeration of ELISPOTs. Weconducted a comparison of individual background (no antigen stimulus) tonative insulin B:9-23, B:9-23 (B22E), and B:9-23 (B21G, 22E) IFN-γresponses in T1D and (B) control subjects. There were more robustresponses to the B:9-23 (B22E) mimotope in both T1D and controlscompared to no antigen and native B:9-23. IL10 responses from T1D andcontrols were also obtained. IL10 ELISPOTs were only measured in asubset of T1D patients (n=8). Overall, controls made more IL10 to theinsulin peptides compared to T1D subjects and have robust IL10 responsesto the B:9-23 (B22E) mimotope. The total IFN-γ ELISPOTs from T1D (n=28)and control subjects (n=27) reveal robust responses to the insulin B22Emimotope (B22Arg→Glu) in comparison to background. Furthermore, theresponses to the insulin B22E mimotope are much greater than that ofnative B:9-23 peptide. We also examined the inflammatory responses basedupon disease status and HLA-DQ genotype. T1D and controls are grouped bythe number of DQ alleles having β57Asp. T1D subjects with two non-B57AspDQ alleles (mainly DQ8 and DQ2) had more IFN-γ producing T cells to theinsulin B22E mimotope compared to HLA matched controls (P=0.02, table2). In individuals having only one DQ allele with non-β57Asp, there wasno difference in IFN-γ ELISPOT responses between T1D and controls forany of the insulin peptides. Among the three insulin peptides, only theB22E mimotope was able to discriminate IFN-γ responses between T1D andcontrol subjects in those having two non-β57Asp DQ alleles. Theresponses to Pentacel, a childhood vaccine, did not differ between T1Dand control subjects (table 3). (Analysis of the data by comparingstimulation index (spot# condition/spot# background) for each peptidedoes not change the response to the insulin B22E mimotope or alter thestatistics).

TABLE 3 Comparison of IFN-γ ELISPOT responses to insulin peptidesbetween T1D and controls based upon DQ Genotype T1D Control P- β57D mean(SEM) mean (SEM) value B: 9-23 −/− 15 (9) 2 (1) ns +/− 2 (1) 2 (1) ns B:9-23 (B22E) −/− 31 (11) 7 (4) 0.02 +/− 29 (17) 35 (11) ns B: 9-23 (B21G,22E) −/− 4 (2) 9 (4) ns +/− 4 (1) 4 (1) ns Pentacel* −/− 244 (25) 247(29) ns +/− 231 (50) 215 (40) ns *Pentacel (positive control) is achildhood vaccine containing immunogens directed against diphtheria,tetanus, pertussis, poliomyelitis, and Haemophilus influenza type b.

With the insulin B22E mimotope providing robust IFN-γ responses, weevaluated the persistence of the response over time in a new-onset T1Dsubject. The B22E mimotope response is reproducible over time withfluctuations in the absolute number of IFN-γ secreting T cells. Duringthis time the subject remained unresponsive to wild type insulin B:9-23and the B21G, 22E mimotope. A positive response to Pentacel was observedfor each ELISPOT assay.

Example 2: Regulatory Responses are Dependent Upon Disease Status andHLA-DQ Genotype

In addition to IFN-γ, IL10 responses were also measured in controls(n=26) and a subset of new-onset T1D patients (n=8). Despite producingsome IFN-γ to the insulin B22E mimotope, controls had significant IL10responses, while several T1D subjects did produce IL10 (FIG. 2).Controls had more IL10 producing cells than diabetics when stimulatedwith native insulin B:9-23 and a trend towards statistical significancewith the B22E mimotope (FIG. 3, left). Examining just the controlsubjects, IL10 producing T cells exist to the insulin B22E mimotope inthose subjects having at least one or both DQ alleles with β57Asp (FIG.3, right). Having this protective polymorphism in the DQ beta chainresulted in 17/18 (94%) controls having greater than 5 IL10 spotscompared to just 3/8 (38%) lacking a protective HLA-DQ allele (p=0.005).

To summarize the cytokine ELISPOT results, cytokine responses to theinsulin B:9-23 (B22E) mimotope are efficiently detected in new-onset T1Dsubjects having DQ alleles associated with diabetes while controlsrespond by dominantly producing IL10 to this peptide when at least onediabetes protective DQ allele is present.

Example 3: Proliferation of CD4 T Cells to the Insulin Peptides

We next examined whether the insulin peptides result in proliferation ofCD4 T cells from the peripheral blood of subjects with established T1Dselected based upon having two non-β57Asp DQ alleles (Table S3).

TABLE S3 Clinical characteristics and HLA genotype of T1D subjects withproliferation assays Case Age T1D DQ HLA DR and DQ Alleles No. (years)Sex (years) Genotype DRB1 DQA1 DQB1 DRB2 DQA2 DQB2 1* 30 F 29 2/8 03010501 0201 0405 0301 0302 2* 32 F 6 2/8 0301 0501 0201 0401 0301 0302 357 F 20 2/2 0301 0501 0201 0301 0501 0201 4* 33 M 20 2/8 0301 0501 02010401 0301 0302 5 23 F 17 8/8 0401 0301 0302 0401 0301 0302 6 28 M 1.52/8 0301 0501 0201 0401 0301 0302 7 28 M 20 2/8 0301 0501 0201 0401 03010302 *T cell receptor Vα gene sequencing performed

PBMCs were isolated from whole blood using Ficoll-paque and resuspendedat a density of 106/ml in CFSE labeling buffer (1% BSA in PBS). Cellswere labeled with 1 μM CFSE (eBioscience) for 10 minutes at 37° C.Labeling was quenched by adding chilled media (IM DM supplemented with5% heat inactivated human AB serum, 100 μg/ml Pen-Strep, 100 μM MEMNEAA, and 50 μM 2-mercaptoethanol) at 5 times the volume at 0° C.; cellswere then incubated on ice for 5 minutes. Labeled cells were washed inPBS with 1% human AB serum, resuspended in media, and plated into a24-well tissue culture plate at 106 cells/well in 1 ml of media.Peptides (Genemed Synthesis Inc.) were HPLC purified (>95%), dissolvedin PBS at a neutral pH, and used at a concentration of 10 μM·DQ antibody(SPV-L3, Abcam) was added at defined concentrations, and Pentacelvaccine (Sanofi Pasteur) was added at 2 μl per well. After seven days ofincubation at 37° C. in 5% CO2, non-adherent cells were harvested andstained for FACS analysis using antibodies to CD4 (RPA-T4, BDBioscience) and CD8 (RPA-T8, BD Bioscience). FACS analysis was doneusing a Becton-Dickenson FACS Caliber and cell sorting for CD4+CFSElocells was done with a Beckman Coulter Moflo XDP 100.

FIGS. 4A-4E show the proliferation results after bulk unfractionatedPBMCs were labeled with CFSE and cultured for 7 days in the presence ofinsulin peptides without the addition of any in vitro stimulus, i.e. noIL-2, anti-CD3, or anti-CD28. Similar to the IFN-γ ELISPOT assays, theB22E mimotope resulted in proliferation of CD4 T cells much more thanthe wild type peptide. Of the tested subjects, all of them had adistinct T cell population proliferating in response to the insulin B22Emimotope more than background and wild type B:9-23 (FIG. 4B, C). Todetermine whether these proliferative responses are DQ restricted, a DQblocking antibody was added to the culture during proliferation. Theantibody is able to block the proliferative response to the mimotope ina dose dependent fashion (FIG. 4D). The DQ antibody doses not block theproliferation of a DR restricted tetanus toxin response, nor does anisotype antibody reduce proliferation (data not shown). All of thetested T1D subjects had less CD4⁺CFSE^(lo) cells following proliferationto the insulin B22E mimotope in the presence of the DQ antibody,indicating the T cell response to the peptide is DQ restricted (FIG.4E).

Example 4: T Cell Receptor V Alpha Gene Usage after Proliferation to anInsulin Peptide

With the ability to proliferate CD4 T cells to the insulin B22Emimotope, we examined whether there is skewing of the T cell receptorvariable (V) genes in CD4⁺CFSE^(lo) cells. In the NOD mouse, it is wellestablished that there is V alpha gene skewing from islet infiltratingCD4 T cells responding to insulin with several dominant V alpha genesused to recognize the insulin B:9-23 peptide (22, 23).

Three individuals had TCR α-chain sequencing performed on sorted CD4 Tcells before peptide proliferation and then on CD4+CFSElo cells afterone week of proliferation. Total RNA was directly extracted from sortedcells using the RNeasy Mini kit (Qiagen) for cells before proliferationand the PicoPure RNA isolation kit (Life Technologies) for those afterproliferation. Single-strand cDNA ligated with the universaloligonucleotide sequence at the 5′ end was synthesized using theClontech SMARTer™ RACE cDNA Amplification Kit according to themanufacturer's instructions. To amplify TCR-α and TCR-β chains, atwo-step PCR was performed. PCR reactions were generated for α- andβ-chains separately using the Universal Primer A Mix supplied from theSMARTer RACE cDNA Amplification Kit along with a primer designed on theconstant region of α-(CCAGGCCACAGCACTGTTGCTCTTGAAGTCC (SEQ ID NO:22))and β-chains (GCTGACCCCACTGTGCACCTCCTTCCC (SEQ ID NO:23)), respectively.The first PCR products were further amplified with nested primersligated with the 454 adaptor sequences containing a multiple identifiersequence: forward primer for both α- and β-chains(CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAAGCAGTGGTATCAACGCAGAGT (SEQ ID NO:24)),reverse primer for α-chains (CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ IDNO:25)—multiple identifier sequence—GCTGGTACACGGCAGGGTCAGGGT(SEQ IDNO:26), and reverse primer for β-chains (CCATCTCATCCCTGCGTGTCTCCGACTCAG(SEQ ID NO:25)—multiple identifier sequence—CACAGCGACCTCGGGTGGGAACAC(SEQ ID NO:27).

These PCR products were agarose gel-purified followed by furtherpurification with the AMPure XP Beads (Beckman Coulter), subject toemulsion PCR with the 454 GSJR titanium chemistry, and sequenced on the454 GSJR instrument (Roche). All sequences obtained from 454 sequencingwere analyzed by the IMGT-HighV-QUEST algorithm (45) to identify Vgene,Jgene, and junction sequences, followed by additional analysis byin-house software to determine frequencies of Vgene usages by individualTCR sequences. Vgene frequencies determined by mean values from the 12PCR reactions were analyzed for individual samples. Alignment clusteranalysis was further performed using the Clustal-Omega algorithm.

In three HLA matched T1D subjects, all having two non-β57Asp DQ alleles,sorted CD4⁺ T cells were analyzed for V gene usage of TCR alpha chainsbefore and after proliferation to the insulin B22E mimotope. There isskewing with several V alpha genes and one V delta gene, located withinthe T cell receptor alpha locus on chromosome 14, used more and lesspredominantly compared to baseline in all three subjects (FIG. 5A).Analyzing phylogenetic trees of Vgene sequences indicates that four ofthe prevalently used V genes (T cell receptor alpha variable [TRAV]38-1, TRAV 38-2, TRAV 19, and T cell receptor delta variable [TRDV] 1)cluster together based upon similarity in CDR1 and CDR2 sequences (FIGS.5B, 5C). The V alpha gene skewing and clustering of dominantly usedgenes based upon CDR1 and CDR2 regions are consistent with antigenspecific T cell proliferation.

The foregoing examples of the present invention have been presented forpurposes of illustration and description. Furthermore, these examplesare not intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with theteachings of the description of the invention, and the skill orknowledge of the relevant art, are within the scope of the presentinvention. The specific embodiments described in the examples providedherein are intended to further explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

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1-42. (canceled)
 43. A method of detecting a predisposition to type 1diabetes mellitus (T1D) in a subject, comprising: contacting T cellsfrom a biological sample from a subject with a peptide comprising theamino acid sequence of SEQ ID NO:2; detecting IFN-gamma and IL10cytokine production by the T cells by enzyme-linked immunospot (ELISPOT)assay; diagnosing the subject as predisposed to T1D when the IFN-gammacytokine production by the T cells from the subject is greater than theIFN-gamma and IL10 cytokine production by T cells from a control sample,and the IL10 cytokine production by the T cells from the subject is lessthan the IL10 cytokine production by T cells from a control sample. 44.The method of claim 43, wherein the subject is diagnosed as predisposedto T1D in the subject when the ratio of IFN-gamma cytokine to IL10cytokine (IFN-gamma/IL10) production by the T cells from the subject isless than the IFN-gamma/IL10 ratio from the control sample.
 45. Themethod of claim 43, further comprising detecting the proliferation ofFoxP3+ regulatory T cells in the biological sample from the subject andcomparing the proliferation in the regulatory T cells to theproliferation in T cells from a control sample.
 46. A method forassessing the efficacy of a therapeutic or preventative therapy in asubject with T1D, or suspected of having T1D, or at risk of developingT1D, comprising: contacting T cells from a first biological sample froma subject with a peptide comprising the amino acid sequence of SEQ IDNO:2 and detecting IFN-gamma and IL10 cytokine production by the T cellsby enzyme-linked immunospot (ELISPOT) assay; contacting T cells from asecond biological sample taken from the subject after a period oftreatment with the therapy, with a peptide comprising the amino acidsequence of SEQ ID NO:2 and detecting IFN-gamma and IL10 cytokineproduction by the T cells by enzyme-linked immunospot (ELISPOT) assay;assessing the therapy as effective when IFN-gamma cytokine production inthe second sample is less than the IFN-gamma cytokine production in thefirst sample; and, assessing the therapy as effective when IL10 cytokineproduction in the second sample is greater than the IFN-gamma cytokineproduction in the first sample.
 47. The method of claim 46, wherein thetherapy is assessed as effective when the ratio of IFN-gamma cytokine toIL10 cytokine (IFN-gamma/IL10) production by the T cells in the secondsample from the subject is greater than the IFN-gamma/IL10 ratio in thefirst sample from the subject.
 48. The method of claim 46, furthercomprising detecting the proliferation of FoxP3+ regulatory T cells inthe first and the second biological samples from the subject andcomparing the proliferation in the regulatory T cells from the secondsample to the first sample.
 49. The method of claim 46, wherein thefirst biological sample and the second biological sample compriseperipheral blood mononuclear cells (PBMC).
 50. The method of claim 46,wherein the therapy comprises administration of a therapeuticallyeffective amount of a peptide selected from SEQ ID NOs:2-21.
 51. Themethod of claim 46, wherein the first biological sample and the secondbiological sample comprise isolated CD4+CD25+ T cells.
 52. A method oftreating type 1 diabetes (T1D) a subject with T1D, or suspected ofhaving T1D, or at risk of developing T1D, comprising administering to asubject in need of such therapy a therapeutically effective amount of acomposition comprising a peptide comprising the amino acid sequence ofSEQ ID NO:2.
 53. The method of claim 52, wherein the composition furthercomprises a therapeutically effective amount of an adjuvant.
 54. Themethod of claim 52, wherein the subject has a mutation at position 57 ofthe of the IA^(g7) beta chain.
 55. The method of claim 54, wherein theamino acid at position 57 of the of the IA^(g7) beta chain is not Asp.